Unit I - Cell Biology Flashcards
Intron splice site consensus sequences
GU marks the 5’ end of an intron
AG marks the 3’ end of an intron
Spliceosome
Made of small nuclear ribonuclear proteins (snRPS) and snRNAs
U1 snRNA contains a sequence complementary to the mRNA sequence near the 5’ splice site of introns
U2 snRNA base pairs with the intron at a point corresponding to the branch point A residue, activating it for nucleophilic attack
Purine accumulation disorders
Defect in hypoxanthine-guanine phosphoribosyl transferase (HGPT); leads to accumulation of purines (uric acid) in tissues
Gout
Lesch-Nyhan Syndrome
Deamination of 5mC and C
Deamination of 5-methyl cytosine converts 5mC to thymine
Deamination of C yields U
Puromycin
Antibiotic; inhibits bacterial translation by mimicking the 3; tRNA acceptor region and interacting with the ribosome to cause premature peptide release
Functions of RNA Poly I, II, III
RNA Polymerase I - synthesizes pre-ribosomal RNA
RNA Polymerase II - synthesizes mRNA
RNA Polymerase III - synthesizes tRNA
TATA Box / Initiator sequence
DNA control element located 25-30bp upstream from the transcription start site; bound by TATa-binding protein (TBP); this is the site at which general TFs bind
TFIID
Contains TATA-binding protein (TBP) as well as TBP-associated factors (TAFs)
Part of RNA Polymerase II pre-initiation complex
Rifampicin
Antibiotic; binds to RNA polymerase at the exit channel and sterically occludes the formation of mRNA chains > 2-3 nucleotides long
TFIIF
Phosphorylates RNA Polymerase II, which activates it
Part of RNA Polymerase II pre-initiation complex
TFIIH
CDK7 subunit phosphorylates Pol II CTD; has some helicase activity and functions in nucleotide excision repair (NER)
Part of RNA Polymerase II pre-initiation complex
Nucleotide Excision Repair (NER)
Removes DNA lesions that distort the DNA structure (i.e. dimers) and therefore block RNA or DNA polymerase from binding
Cockayne Syndrome & Xeroderma Pigmentosum are caused by defects in NER, often TFIIH
PolyA tail cleavage site consensus sequence
AAUAAA 10 to 30 nucleotides upstream from the cleavage site
DNA Polymerase III
The main polymerase involved in DNA replication; Pol III “holds on” to the DNA via a sliding clamp protein and has 3-5’ exonuclease activity that enables proofreading
DNA Polymerase I
Mostly involved in replacing RNA primer with DNA; has exonuclease activity in the 5-3; direction
DNA Mismatch repair
MutS/MutL (in prokaryotes) or MSH/LH (in eukaryotes) recognize mismatched DNA nucleotides on the newly synthesized strand, which is unmethylated
Defects in this pathway cause hereditary non-polyposis colorectal cancer (HNPCC)/Lynch Syndrome
Base Excision Repair (BER)
Removes DNA lesions that do not cause distortions in the backbone and therefore may be missed by NER; glycosylase enzymes hydrolyze the N-glycosidic bond to remove the damaged base, producing an AP site which is removed by AP-specific endonuclease
Homologous Recombination & Non-homologous end joining (NHEJ)
Both repair double-stranded DNA breaks; HR repairs breaks between homologous ends; it requires the presence of a homologous chromosome (S & G2 phase)
NHEJ repairs breaks between non-homologous ends, which may lead to insertion/deletion of nucleotides at the break point
4 categories of DNA binding domains
Helix-Turn-Helix (Homeodomain)
Zinc Finger
Basic Leucine Zipper Proteins (bZLP)
Basic Helix-Loop-Helix motif (bHLH)
Histone structure
Histones are octameric proteins consisting of dimers of subunits H2A, H2B, H3, and H4
Histone H1 is bound to linker DNA between histones and functions to “slide” histones along DNA during modification
Histone De/Acetylation
Histone Acetyltransferases (HATs) and Histone De-acetyltransferases (HDATs) are classes of reversible chromatin modifiers; acetylation of histone N-termini Lysine residues neutralizes the positive charge and “frees” the histone tails from electrostatic reactions with the DNA phosphate backbone
Stop Codons
UAA
UAG
UGA
Shine-Delgarno Sequence
Prokaryotic ribosomal binding site; essentially corresponds with initiator AUG codon
Initiation Factor 4E (IF4E)
Binds the 7-methyl guanosine cap on the 5’ end of the mRNA, facilitating scanning of the ribosome down the primary transcript to the start AUG codon
Elongation Factors (Prok and Euk)
Ef-Tu (in bacteria) & Ef1A (in eukaryotes); deliver charged aa-tRNA complexes to the A site of the ribosome, burning GTP when it dissociates
Initiation of Translation - Bacteria
IF1 and IF3 bind the small subunit, which binds the mRNA at the AUG start codon via base pairing with the Shine Delgarno sequence
IF2 (bound to GTP) delivers initiation Met-tRNA to the P site to pair with the AUG codon
GTP hydrolysis on IF2 releases all IFs and signals binding of the large subunit
DNA Polymerase delta
Performs DNA synthesis on the lagging strand in eukaryotes; contains 3-5’ exonuclease activity
DNA Polymerase alpha
Functions in the synthesis of the first ~20 DNA nucleotides immediately following the RNA primers; also synthesizes the first basepairs of each Okazaki fragment
DNA polymerase alpha has primase activity
DNA Polymerase epsilon
Performs DNA synthesis on the leading strand in eukaryotes; contains 3-5’ exonuclease activity
Release Factor (RF)
Enters the A site (bound to GTP) in conjunction with a stop codon; hydrolysis of GTP induces release of the ribosomal subunits
Sources & Types of High Energy Bonds
Thioester bonds (i.e. Acetyl coA) Phosphoanydride bonds (i.e. ATP) Phosphoenolpyruvate (PEP)
Relationship between free energy, enthalpy, and entropy of a system
dG = dH - TdS
Reduction Potential (E)
A measure of the willingness of a molecule to become reduced; the more positive the value of E, the more likely a molecule is to accept electrons (become reduced); the more negative the value of E, the more likely a molecule is to donate electrons (become oxidized)
Energy is released as electrons pass from compounds with low E to high E values
Relationship between free energy and redox potential
dG = -nFdE
Enzyme cofactor vs. coenzyme
Cofactors are small molecules (often metal ions) that function with an enzyme to catalyze a reaction; cofactors are not used up in the reaction
Coenzymes provide chemical groups for a catalyzed reaction; they are used up by the reaction
Prosthetic groups are cofactors/coenzymes that are tightly bound to the enzyme
Km
Km is the substrate concentration at which Vo = 1/2(Vmax)
Km typically approximates intracellular concentrations of [S]
Kcat
Kcat describes the number of substrate molecules turned into product per enzyme molecule, per second, at saturating substrate concentrations
The larger the Kcat value, the faster the enzyme works to turnover
Kcat/Km
A measure comparing enzyme efficiency; the larger the value of Kcat/Km, the more efficient the enzyme
Enzymes that are “catalytically perfect” have a Kcat value approaching 10^8; the enzyme is so efficient that rate of diffusion is the only limiting factor
Inhibitors - 3 classifications
Competitive - Bind to the enzyme active site, blocking binding of the natural substrate
Uncompetitive - Bind to the ES complex at a site other than the active site
Mixed - May bind to either E or ES at a site other than the active site
Alpha Helix
H-bonds form between the carbonyl oxygen of the nth AA and the amide nitrogen of the n + 4th AA within the same polypeptide chain, forming a right-handed screw
Ala and Leu are commonly found; Pro and Gly do not occur
Beta Sheets
Secondary structure motif formed by hydrogen bonds between two polypeptide chains running parallel or anti-parallel to each other
Ex: Immunoglobulin, antibodies
Kd
Dissociation constant; Kd is the ligand concentration at which 50% of the protein is ligand-bound
Smaller Kd value implies a stronger binding interaction
Myoglobin
Stores oxygen; oxygen binds Fe2+ in the heme group, which is buried within the myoglobin protein; Kd is very low, so myoglobin stores oxygen but does not transport
Hemoglobin
Hb has 4 O2 binding sites that interact with allostery; O2 binding triggers a T to R conformational state change in which Hb has higher affinity for O2
Hb binds O2 better under local conditions of higher pH (i.e. in the lungs) and releases O2 under conditions of lower pH (i.e. in metabolically active tissues)
2 Classes of Chaperone Proteins
- HSP70 - binds to the hydrophobic regions of unfolded proteins, preventing aggregation and facilitating re-folding
- GroEl/GroES (prok) / HSP60 (euks) - forms a capped, barrel-like structure that isolates mis-folded proteins and hydrolyzes ATP to aid in re-folding
Alzheimers - 3 primary genetic pathways
- Missense mutation in APP gene causes increased production of all B-amyloid proteins, including B-42 via cleavage by B-secretase and y-secretase
- ApoE4 genetic variant leads to decreased AB 42 clearance
- Mutation in presenelin genes, leading to increased production of AB42 (more common in familial cases of Alzheimers)
Stages of the Cell Cycle
G1 - Cell growth; building and loading of Pre-RC onto origin
S - Activation of Pre-Rc, DNA Synthesis
G2 - Protein synthesis and cell growth
M - Chromosome Segregation
Rad 17, ATM/ATR, p53, p21, Rb cycle
Rad17 senses & binds to damaged DNA, recruiting ATM/ATR
ATM/ATR proteins kinases phosphorylate p53
Phosphorylated p53 is a TF that promotes synthesis of p21, a CDK inhibitor
p21 inhibits CDK, which fails to phosphorylate Rb
Unphosphorylated Rb binds to and inhibits transcription factor E2F, which is necessary for transcription of a group of genes involved in DNA synthesis
In response to DNA damage the cell is held in G1 phase and cannot progress to S phase
Pre-RC Cycle
Pre-RC is built and loaded onto replication origins in G1; Pre-RC are activated during S phase
This ensures that cells may not replicate and divide during the same phase of the cell cycle
VDJ Recombination
An endogenous source of double stranded DNA breaks responsible for generation of diversity in immunoglobulin DNA via non-homologous end joining; mutations in NHEJ genes lead to severe combined immunodeficiency (SCID) because functional B-cells and T-cells are not produced
Crohn’s Disease
Most often affects the Ileum & Colon, although may affect the entire GI tract
Characterized by: discontinuous lesions, transmural inflammation, perianal disease, strictures & fistulas
Ulcerative Colitis
Most often affects the colon & rectum
Characterized by: continuous lesions, mucosal inflammation, hematochezia
Extra-GI manifestations of Crohn’s / Ulcerative Colitis
Infections of the eyes - Iritis, episcleritis
Infections of the lungs & heart - pleuritis, myocarditis
Pancreatitis
Ankylosing Spondylitis (chronic inflammatory arthritis)
Sacroileitis
Erythema Nodosum - inflammation of subcutaneous fat (usually on shins)
Pyoderma gangrenosum (chronic ulcers on the legs)
Swi/Snf
Chromatin remodeling complex that uses DNA to break histone/DNA contacts and slide the histones down the DNA, “loosening” the chromatin structure
Tetracycline, Streptomycin, Erythromycin
Antibiotic drugs that bind to the small subunit of the prokaryotic ribosome, preventing entry of aa-tRNAs into the A site and thus inhibiting bacterial protein synthesis
Interferon
Interferon is released by cells in response to viral infection; in neighboring cells, it stimulates the activity of kinases which phosphosrylates eIF2; when phosphorylated, eIF2 is bound by eIF2-BP and initiation of translation is stalled
eIF2
A eukaryotic initiation factor required for translation; eIF2 mediates the binding of the initiator t-Met complex to the ribosomal P site; after formation of the proper codon-anticodon pairing, eIF2 hydrolyzes GTP and releases from the ribosome
Ciclosporin
An immunosuppressant drug; ciclosporin binds to cyclophilin, inhibiting activation of calcineurin; normally, calcineurin functions to dephosphorylate the transcription factor NFAT, allowing it to move to the nucleus and activate transcription of genes coding for immune cell proliferation; ciclosporin therefore prevents translocation of the TF NFAT
Rubenstein-Taybi Syndrome
Results from mutations in one copy of the CREB-BP gene; CREB-BP is a transcriptional co-activator for many TFs and is a HAT; presents as cognitive disability & craniofacial dysmorphism
a-aminitin toxin
Death cap mushroom toxin; blocks the translocation of RNA polymerase II by interacting with its bridge helix
Tamoxifen
Tamoxifen antagonizes estrogen by binding to the estrogen receptor (ER) and preventing recruitment of HAT co-factors (activators)
NFKB Pathway
NFKB protein is usually held in the cytoplasm by binding to its inhibitor (IKB); phosphorylation of IKB targets it for degradation, releasing NFKB to move into the nucleus where it turns on its target genes, including genes involved in inflammation
Aspirin inhibits the phosphorylation of IKB
Rapamycin
An immunosuppressant drug; Inhibits mTOR (mammalian target of Rapamycin), which is a kinase that phosphorylates 4E-BP; inhibition of mTOR leads to dephosphorylation of 4E-BP, which is the state in which it binds eIF4E, silencing translation
